The present invention relates generally to the fields of biology and chemistry. In particular, the present invention is directed to compositions and methods for use in voltage sensing, especially in biological systems.
Fluorescence detection and imaging of cellular electrical activity is a technique of great importance and potential [Grinvald, A., Frostig, R. D., Lieke, E., and Hildesheim, R. 1988. Optical imaging of neuronal activity. Physiol. Rev. 68:1285-1366; Salzberg, B. M. 1983. Optical recording of electrical activity in neurons using molecular probes. In Current Methods in Cellular Neurobiology. J. L. Barker, editor. Wiley, New York. 139-187; Cohen, L. B. and S. Lesher. 1985. Optical monitoring of membrane potential: methods of multisite optical measurement. In Optical Methods in Cell Physiology. P. de Weer and B. M. Salzberg, editors. Wiley, New York. 71-99]. Mechanisms for optical sensing of membrane potential have traditionally been divided into two classes: sensitive but slow redistribution of permeant ions from extracellular medium into the cell, and fast but small perturbations of relatively impermeable dyes attached to one face of the plasma membrane [Loew, L. M. 1988. How to choose a potentiometric membrane probe. In Spectroscopic Membrane Probes. L. M. Loew, editor. CRC Press, Boca Raton. 139-151; Loew, L. M. 1993. Potentiometric membrane dyes. In Fluorescent and Luminescent Probes for Biological Activity. W. T. Mason, editor. Academic, San Diego. 150-160].
The permeant ions are sensitive because the ratio of their concentrations between the inside and outside of the cell can change by up to the Nernstian limit of 10-fold for 60 mV change in transmembrane potential. Their responses are slow because to establish new equilibria, ions must diffuse. through unstirred layers in each aqueous phase and the low-dielectric-constant interior of the plasma membrane.
By contrast, the impermeable dyes can respond very quickly because they need little or no translocation. They are insensitive, however, because they sense the electric field with only a part of a unit charge moving less than the length of the molecule, which in turn is only a small fraction of the distance across the membrane. Furthermore, a significant fraction of the total dye signal comes from molecules that sit on irrelevant membranes or cells and that dilute the signal from the few correctly placed molecules.
It is an object of the present invention to provide compositions and methods which do not suffer from all of the drawbacks of the prior art.